3. Remove a cover-slip from the pot of absolute alcohol with sterile forceps; burn off the alcohol in the gas flame. 4. Lower the now sterile cover-slip carefully on to the inoculated surface of the medium, carefully excluding air bubbles, and press it down firmly with the points of the forceps. (A sterile disc of mica may be substituted for the cover-slip.) 5. Incubate. ~Method III~ (Roux's Physical Method).-- 1. Prepare tube cultures of fluid media (or solid media rendered fluid by heat) in the usual way. 2. Aspirate some of the inoculated media into capillary pipettes. 3. Seal both ends of each pipette in the blowpipe flame. 4. Incubate. ~Method IV~ (Roux's Biological Method).-- 1. Plant a deep stab, as in method I. 2. Pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous aerobe--e. g., B. aquatilis sulcatus or B. prodigiosus--upon the surface of the medium; or an equal depth of liquefied gelatine, which is then inoculated with the aerobic organism. 3. Incubate. The growth of the aerobe will use up all the oxygen that reaches it and will not allow any to pass through to the medium below, which will consequently remain in an anaerobic condition. (B) ~Method V.~-- 1. Prepare tube or flask cultivations in the usual way. 2. Replace the cotton-wool plug by an india-rubber stopper perforated with one hole and fitted with a length of glass tubing which has a constriction about 3 cm. above the stopper and is then bent at right angles (Fig. 129). The stopper and glass tubing are sterilised by being boiled in a beaker of water for five minutes. [Illustration: FIG. 129.--Vacuum culture.] 3. Connect the tube leading from the culture vessel with a water or air pump, interposing a Wulff's bottle fitted as a wash-bottle and containing sulphuric acid. 4. Exhaust the air from the culture vessel. 5. Before disconnecting the apparatus, seal the glass tube from the culture vessel at the constriction, using the blowpipe flame. 6. Incubate. (C) ~Method VI~ (Buchner's Method). ~Apparatus and Solutions Required.~-- Buchner's tube (a stout glass test-tube 23 cm. long and 4 cm. in diameter, fitted with india-rubber stopper, Fig. 130). Pyrogallic acid in compressed tablets each containing 1 gram. Dekanormal solution of caustic soda. METHOD.-- 1. Prepare the tube cultivation in the usual way. 2. Moisten the india-rubber stopper of the Buchner's tube with water and see that it fits the mouth of the tube accurately. 3. Remove the stopper from the caustic soda bottle. 4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube (roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity of the receiving vessel). 5. Add about 1 c.c. of the soda solution. 6. Place the inoculated tube inside the Buchner's tube. The pyrogallic tablet acts as a buffer and prevents damage to either the inoculated tube or the Buchner's tube even should it be slipped in hurriedly.